+

WO1993000064A2 - Evaluation et traitement du phenotype de resistance multimedicamenteuse - Google Patents

Evaluation et traitement du phenotype de resistance multimedicamenteuse Download PDF

Info

Publication number
WO1993000064A2
WO1993000064A2 PCT/US1992/005329 US9205329W WO9300064A2 WO 1993000064 A2 WO1993000064 A2 WO 1993000064A2 US 9205329 W US9205329 W US 9205329W WO 9300064 A2 WO9300064 A2 WO 9300064A2
Authority
WO
WIPO (PCT)
Prior art keywords
cell
compound
multidrug resistance
detected
cells
Prior art date
Application number
PCT/US1992/005329
Other languages
English (en)
Other versions
WO1993000064A3 (fr
Inventor
David R. Piwnica-Worms
Original Assignee
Brigham And Women's Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Brigham And Women's Hospital filed Critical Brigham And Women's Hospital
Publication of WO1993000064A2 publication Critical patent/WO1993000064A2/fr
Publication of WO1993000064A3 publication Critical patent/WO1993000064A3/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
    • A61K51/0476—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group complexes from monodendate ligands, e.g. sestamibi
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/13—Tracers or tags

Definitions

  • the present invention relates to methods for detecting the multidrug resistance phenotype in vivo and in vitro.
  • the invention particularly relates to methods of diagnosing the multidrug resistance pheno ⁇ type by imaging, particularly scintigraphic imaging, in solid tumors in vivo or in tumors and biopsies in vitro.
  • the methods of the present invention allow the diagnosis of multidrug-resistant tumors and other multidrug-resistant phenotypes without invasive surgical methods.
  • the invention also relates to methods to increase the net uptake of drugs, and especially chemotherapeutic drugs, into cells and particularly to cells in vivo in malignant tumors.
  • the invention is directed to new chemosensitizing agents and their use to enhance the uptake of various chemotherapeutic drugs.
  • the methods of the present invention allow therapy of patients with multidrug resistant tumors.
  • the methods of therapy of the present invention use compounds that interact with the multidrug resistance transport protein.
  • the invention is directed to multidrug-resistance reversal agents that inhibit the cellular efflux of chemotherapeutic and other cytotoxic drugs in vivo and in vitro.
  • the conserved domain comprises nearly the entire polypeptide. In others the conserved domain is only one segment of a multi-domain protein.
  • These proteins include the multidrug resistance P- glycoprotein, the product of the White locus of Drosophila, procaryotic proteins associated with membrane transport, cell division, nodulation and DNA repair, the STE-6 gene product that mediates export of yeast ⁇ -factor mating pheromone, pfMDR that is implicated in chloroquine resistance of the malarial parasite, and the product of the cystic fibrosis gene (CFTR) .
  • ATP binding proteins There are two short amino acid sequence motifs which are present in most if not all nucleotide binding proteins.
  • the subfamily of ATP binding proteins that are relevant to this invention are distinct from all other ATP binding proteins in that they share considerably more sequence identity than is simply required for nucleotide binding. Other ATP binding proteins may possess the consensus nucleotide binding motifs but otherwise share no significant sequence similarity. This implies that the subfamily of proteins shares common functions in addition to the ability to bind ATP.
  • Many of the proteins of the subfamily and those which are best characterized are components of an active transport system hich mediates the transport of molecules across the cyto ⁇ plasmic membrane. They are recognized in the art as ATP-binding cassette superfamily of transport proteins (Hyde, S., et al . , Nature 346: 362 (1990)).
  • nucleotide binding protein-dependent transfer systems have been characterized in procaryotes. Each system requires a substrate binding protein located in the periplas that provides a primary receptor for transport. The system also contains two integral membrane proteins that transport substrates across the membrane. The system further contains two peripheral membrane proteins thought to be located on the inner surface of the cytoplasmic membrane. These peripheral membrane proteins are members of the subfamily of bacterial and eucaryotic
  • P-glycoprotein is a eucaryotic four-domain protein consisting of two hydrophobic domains and tv ATP binding dom is . Besides the conserved ATP binding domains, there is a great deal of similarity between P-glycoprotein and bacterial binding protein dependent transport systems. The organization of this protein is remarkably similar to that of bacterial transport systems. The two hydrophobic domains in P-glycoprotein are homologous to each other. The same is true for the two hydrophobic components of the binding protein system.
  • the P-glycoprotein consists of four domains encoded as a single polypeptide, whereas in bacteria the equiva ⁇ lent domains are on separate polypeptides.
  • P-glycoprotein pumps drugs out of the cell whereas the binding protein dependent transport system mediates uptake.
  • protein dependent transport systems require a periplasmic component which serves as the initial substrate binding site and delivers substrate to the membrane component.
  • periplasmic component which serves as the initial substrate binding site and delivers substrate to the membrane component.
  • P-glycoprotein exhibits a very broad speci ⁇ ficity, handling a range of apparently unrelated drugs.
  • most of the differences may be trivial rather than fundamental mechanistic differ ⁇ ences. It is not uncommon for functions carried out by separate polypeptide chains in procaryotes to be fused into a single multifunctional protein in eukaryotes.
  • a small change in the organiza ⁇ tion of a transport system could promote efflux rather than uptake.
  • the periplasmic components of bacterial systems can be viewed as a specific adapta ⁇ tion to the fact that bacteria have a periplasm.
  • P-glycoprotein The similarity between P-glycoprotein and the bacterial active transport system may be relevant to the mechanisms of multidrug resistance in eucaryotic cells. All available evidence is compatible with the view that P-glycoprotein is a eucaryotic transport system. Most chemotherapeutic drugs are lipophilic and can enter the cells passively. In multidrug- resistant cells, the intracellular concentration of these drugs is reduced in an energy-dependent manner. The most reasonable explanation for these findings is that P-glycoprotein is an active transport system, pumping drugs out of the cell.
  • Tissue culture eel ? can be selected for resistance to a variety of drugs such as colchicine, doxorubicin (Adriamycin) , actinomycin D and vinblastine. Increas ⁇ ing the concentration of the selecting agent in multiple small single steps results in high levels of cross resistance to these agents as well as many other drugs including other anthracyclines, Vinca alkaloids and epipodophyllotoxins (Gottesman, M.M. et al . , J. Biol . Chem . 263 : 12163 (1988)).
  • drugs such as colchicine, doxorubicin (Adriamycin) , actinomycin D and vinblastine. Increas ⁇ ing the concentration of the selecting agent in multiple small single steps results in high levels of cross resistance to these agents as well as many other drugs including other anthracyclines, Vinca alkaloids and epipodophyllotoxins (Gottesman, M.M. et al . , J.
  • Cells or tissues obtained from tumors and grown in the presence of a selecting cytotoxic drug can result in cross-resist ⁇ ance to other drugs in that class as well as other classes of drugs including anthracyclines, Vinca alkaloids, and epipodophyllotoxins (Riordan et al . , Pharmacol . Ther.
  • the characteristics of the multidrug resistance phenotype have been analyzed by studies on normal and tumor cell lines isolated for resistance to selected cytotoxic drugs.
  • One major mechanism of multidrug resistance in mammalian cells involves the increased expression of the 170-kDa plasma membrane glyco- protein, P-glycoprotein (for review, Juranka et al . , FASEB J 3:2583 (1989); Bradley, G. et al . , Biochem. Biophys. Acta 948 : 87 (1988)).
  • Transfection of cloned P-glycoprotein genes into drug-sensitive cell lines has confirmed that an increased expression of P- glycoprotein is sufficient to cause multidrug resist ⁇ ance in experimental systems (i.e., Gros, P. et al .
  • the nucleotide sequence of multidrug resistance cDNA indicates that it encodes a polypeptide similar or identical to P-glycoprotein and that these are members of the highly conserved class of membrane proteins similar to bacterial transporters and involved in normal physiological transport processes.
  • the multidrug resistance P-glycoprotein may function normally to extrude as yet unknown physiol- ogical substrates out of cells by an energy-dependent process (Arceci, R.J. et al . , PNAS USA 85:4350 (1988)) in normal tissues.
  • the gene is amplified and con ⁇ sequently overexpressed in malignant tissues. It is thus believed that by transporting chemotherapeutic agents out of the cells, P-glycoprotein renders tumors resistant to chemotherapy.
  • Multidrug resistance has been detected in vitro in single cell suspensions and in cell monolayers.
  • Yoshimura et al . (Cancer Letters 50:45 (1990)) used the uptake of rhodamine dye to screen for agents that overcome multidrug resistance in a cell line ("revers ⁇ ing agents").
  • the dye is accumulated in multidrug-resistant cells at a lower rate than it is accumulated in non-resistant cells and thus multidrug-resistant cells can be distinguished from non-resistant cells by comparing intracellular dye levels.
  • the authors monitored dye levels in multidrug-resistant cells in the presence and absence of verapamil, a known chemosensitizer (revers ⁇ ing agent used in chemotherapy to facilitate the uptake of a chemotherapeutic drug in drug-resistant tumor cells) , and found that the dye accumulated to normal levels when the multidrug resistance phenotype was reversed with verapamil.
  • the dye was administered to cells in a confluent monolayer. The cells were then either washed, solubilized, and the dye detected with a fluorescence spectrometer, or scanned in microtitre wells with a fluorescence microplate reader.
  • E ⁇ ferth et al . (Arzneim-Forsch 38:1771 (1988)) also developed an in vitro assay to detect the multi- drug resistance phenotype. They compared the levels of rhodamine dye in a cell sample with the levels of dye found in a control sample of normal cells. The dye was detected by forming a single cell suspension, pipetting the suspension onto slides, administering the dye to the cells on the slide and detecting dye uptake of cells on the slide.
  • Herweijer et al . (Invest New Drugs 7:442 (1989)) used on-line flow cytometry to detect cells with the multidrug resistance phenotype in a single cell suspension.
  • the uptake kinetics of a fluorescent drug were measured on line first in the absence and then in the presence of a reversing agent. "**"
  • Hexakis (R-isonitrile) technetium (I) complexes are a class of low valence technetium ( 59m Tc) coordination compounds empirically designed as clinical myocardial perfusion imaging agents (Jones, A.G. et al . , Int . J. Nucl . Med . Biol . 11 : 225 (1984), Holman, B.L. , et al . , J. Nucl . Med. 25:1350 (1984), Holman, B.L., et al . , ibid 28:13 (1987), Sporn, V., Clin.
  • the ter ⁇ minal R groups when bound to the technetium, encase the metal with a sphere of lipophilicity (Jones, A.G., et al . , Int . J. Nuc . Med . Biol . 11:225 (1984), Mousa, S.A., et al . , J. Nuc Med . 28:1351 (1987)).
  • P-glycoprotein function Based on the information obtained from amino acid sequence analysis of P-glycoprotein from various mammalian cells, a model for P-glycoprotein function has been suggested (Bradley et al . , Biochimica et Biophysica Acta 948 : 87 (1988)). The model suggests that P-glycoprotein forms a channel in the plasma membrane and transports drugs out of cells using energy derived from ATP hydrolysis. In one version of the model, P-glycoprotein binds drugs directly and then removes them from the cell. It is suggested that, since transfection of a P-glycoprotein cDNA clone into drug sensitive cells results in cross- resistance to structurally unrelated drugs, the P-glycoprotein molecule may have binding sites for a diverse group of drugs.
  • the 150-180 kilodalton protein that binds vinblastine was immunoprecipitated with a monoclonal antibody against P-glycoprotein (Cornwell, M.M. , et al . , J. Biol . Chem . 262 : 2166 (1987)).
  • a drug binding protein is transported out of cells by a P-glycoprotein pump. Drugs may bind irreversibly to this protein, and the entire drug-protein complex may then be removed from the cell. Direct binding of drug analogs to the
  • P-glycoprotein has been observed.
  • P-glycoprotein can be labeled directly by a photoactive vinblastine analog in a saturable manner. This photoaffinity labeling can be inhibited by drugs such as daunomycin or vincristine, as well as several chemosensitizing agents such as verapamil, quinidine, reserpine, and azidopine (Gottesman, M.M., et al . , Trends Pharmacol . Sci. 9: 54 (1988)).
  • the labeling of P-glycoprotein by a photoactive analog of verapamil can be inhibited by some, but not all, drugs involved in the MDR phenotype (Safa, A.R., Proc. Natl . Acad. Sci. USA 85: 7187 (1988)). Because these drugs and reversing agents may inhibit binding to P-glycoprotein, this suggests that a common binding site may be involved.
  • a mechanism of reversal of the MDR phenotype by reversing agents/chemosensitizers may be explained on the basis of competition for drug binding, which results in decreased efflux of drugs which are taken up by the cell and thus a higher intracellular level of such drugs, such as chemotherapeutic drugs, in cells that are multidrug resistant.
  • the chemosensitizers described to date may be grouped into six broad categories: (1) calcium channel blockers; (2) calmodulin antagonists; (3) non- cytotoxic anthracycline and Vinca alkaloid analogs; (4) steroids and hormonal analogs; (5) miscellaneous hydrophobic cationic compounds; and (6) cyclosporines. Although these compounds share only broad structural similarities, most are extremely lipophilic, and those in the first five groups are all heterocyclic, amphipathic substances (Ford, J.M. , et al . , Pharmacol . Rev. 42:155 (1990)).
  • P-glycoprotein With respect to human malignancy, an important question is the physiologic relevance of P-glycoprotein. Specific questions are whether multidrug resistance based on P-glycoprotein overexpression occurs in malignancy, and if it does occur, what is its relation to the success of chemotherapeutic treatment. Increased levels of P-glycoprotein have b-en seen in some late stage ovarian carcinomas (B xl, D.R. , et al . , J. Ciin . Oncol . 3:311 (1985)).
  • P-glycoprotein or P-glycoprotein messenger RNA have been detected in all forms of human cancers, including leukemias, lymphomas, sarcomas, and carcinomas (Goldstein, L.J., et al . , J. Natl . Cane . Inst . 81:116 (1989) ) .
  • An increased level of P- lycoprotein was observed in tumor biopsies obtains... af* sr relapse during chemotherapy, compared with tumor biopsies obtained before the treatment.
  • relatively high levels of P-glycoprotein were seen in some tumors even before chemotherapy.
  • P-glycoprotein occurs in human malignancy.
  • the relationship to the response to chemotherapy is.not yet clear.
  • Chemosensitizers appear to act by directly affecting the function of P-glycoprotein. For example, labeled vinblastine accumulated in P-glycoprotein enriched membrane vesicles in multidrug resistant cells in a specific saturable temperature- dependent manner not observed in vesicles from either drug sensitive cells or multidrug resistant revertants to sensitivity. Accumulation could be inhibited by excess unlabeled vinblastine, vincristine and verapamil (Cornwell, M.M. , et al . , J. Biol . Chem . 261:7921 (1986)).
  • chemosensitizers have been shown to bind to membranes enriched for P-glycoprotein, and this binding was inhibited by other chemosensitizers and by chemotherapeutic drugs (Ford, J.M., et al . , Pharmacol . Rev. 42:155 (1990)).
  • Many studies have shown that chemosensitizers may be substrates for the P-glycoprotein transport system, supporting the hypothesis that the mechanism of chemosensitization is as a competitive ligand for a site on the P-glycoprotein (Ford, J.M. , et al . , Pharmacol . Rev. 42 : 155 (1990)).
  • the present invention is based on the unexpected discovery that the lipophilic cationic gamma-emitting imaging agent, hexakis (2-methoxyisobutyl isonitrile) technetium-99m (I) (Tc-MIBI), is transported out of cells against a concentration gradient by P- glycoprotein, the product of the multidrug-resistance gene and a member of the family of ATP-binding cassette transport proteins.
  • Tc-MIBI hexakis (2-methoxyisobutyl isonitrile) technetium-99m
  • living cells are imaged.
  • Agents that are useful in imaging procedures are administered to a patient or a tissue specimen. Imaging procedures include, but are not limited to, magnetic resonance, superconducting quantum interference device (squid) , positron emission tomography, and, in highly preferred embodiments, imaging is by planar scintigraphy or single photon emission computed tomography (SPECT) .
  • SPECT single photon emission computed tomography
  • the method is applicable as a rapid and simple assay of multidrug-resistant cells in vitro and, more importantly, as an assay in instances in which pres ⁇ ently available assay methods are impractical or impossible.
  • multi ⁇ drug-resistant cells are detected without the need for tissue dispersion and growth that could change the in vivo phenotype.
  • the method is especially valuable as an in vivo assay whereby multidrug resistance tissue is detected without the need for traumatic surgery.
  • multidrug-resistant tumors are detected in cancer patients without the need for surgery.
  • the multidrug resistance phenotype is detected with Tc-MIBI.
  • Tc-MIBI is without chemotoxic effects.
  • detection is with the broader range of technetium complexes, other non-toxic imaging agents, and other non-toxic markers that are transported by the multidrug resistance transport system and which can be detected despite the occurrence of biological tissue intervening between the cells and the imaging device.
  • the invention is directed to methods of detecting the multidrug resistance phenotype in an animal, tissues, or cells by administering an agent which is transported by the multidrug resistance transport system and which is detectable in living cells, at distances removed from the cells by the presence of intervening tissue.
  • the invention is particularly directed to methods of detecting the multidrug resistance phenotype in an animal, tissues, or cells by administering an imaging agent, especially a scintigraphic imaging agent, which is transported by the multidrug resistance transport system.
  • the invention is particularly directed to a method of assaying the multidrug resistance phenotype of solid tumors in vivo and in vitro by administering to patients, explanted tumor, or cells, an agent that is transported by the multidrug resistance transport system.
  • the invention is also directed to designing chemotherapy regimens by assaying the multidrug resistance phenotype in patients or their explanted tissue either prior to or during treatment.
  • the agent alone is administered to the subject (cells, tissue, or patient) and the incorporation of agent is measured. Thereafter, the agent is co-administered with a reversing agent and the incorporation of the agent is again measured. If the subject contains multidrug-resistant cells, these cells will accumulate less of the agent when the agent alone is administered than they will when the agent is administered with a reversing agent. Thus, when the two measurements are compared, greater intracellular accumulation of the agent in the presence of the reversing agent indicates the presence of multidrug-resistant ceils.
  • the invention is also practiced with any of the family of ATP-binding transport proteins of which the multidrug resistance transport protein P-glycoprotein is a member.
  • the present invention based on the unexpected discovery that Tc-MIBI is transported out of cells against a concentration gradient by P-glycoprotein, is now extended to therapy exploiting a wide variety of hexakis (alkylisonitrile) metal and hexakis (arylisonitrile) metal complexes. These hexakis metal isonitrile complexes define a new class of multidrug resistance reversal agents and direct chemotherapeutic agents useful in chemotherapeutic protocols.
  • the present invention describes the structure of metal isonitrile complexes that are designed to have a high affinity for the multidrug resistance transport protein and, as such, be potent reversal agents by inhibiting the cellular efflux of chemotherapeutic and cytotoxic drugs, as well as being chemotherapeutic agents per se.
  • the present invention is also directed to methods for reversing the multidrug resistance phenotype in cells comprising administering the complexes of the present invention to cells with the multidrug resistance phenotype.
  • the administration may be in vitro or in vivo.
  • the complexes are administered to cancer patients in vivo as a means of treating tumors which have become multidrug resistant.
  • the complexes are designed to be cytotoxic and are administered alone. In more preferred embodiments of the invention, the complexes are coadministered with a chemotherapeutic drug. In this aspect, the complexes of the present invention act as a chemosensitizer or reversing agent causing the enhanced accumulation of the chemotherapeutic drug as a result of the reversal of the multidrug resistance phenotype.
  • the complexes of the present invention may also be used in vitro to study cytotoxicity in screening protocols for new cytotoxic compounds or in tissue biopsies from cancer patients to determine effective cytotoxic agents for a particular patient.
  • the complexes can be synthesized with both stable and radioactive core metals.
  • typical structures are hexakis isonitrile complexes of a core metal which may be, but is not limited to, rhenium or technetium.
  • multiligand isonitrile complexes of copper, iron, cobalt, manganese, ruthenium, platinum, osmium, iridium, tungsten, chromium, molybdenum, nickel, rhodium, palladium, niobium and tantalum can also be synthesized.
  • all metals but technetium have stable (non-radioactive) isotopes found in nature and which are suitable for drug manu acture.
  • radioactive isotopes of all the metals may be synthesized for use during drug design, for example, for performing biodistributions or for therapeutic exploitation of the radioactive emissions into cancer tissues.
  • the side groups of these compounds are substituted alkyl and aryl isonitrile ligands.
  • FIG. 1 Accumulation of Tc-MIBI in LZ cells (highly expressing multidrug resistance) (0) and V79 cells (modestly expressing multidrug resistance) (•) .
  • Cells were incubated in modified Earle's balanced salt solution (MEBSS) containing 99 Tc-MIBI (53 uCi/ml; 20.7 pmol/mCi) for various times and cell associated activity determined as described previously (Chiu et al . , J. Nucl . Med. 31:1646-1653 (1990)). Results are expressed as fmol cellular Tc-MIBI/mg protein per nmolar extracellular Tc-MIBI. Points represent the mean + SEM of three determinations each.
  • FIG. 3 Inhibition of Tc-MIBI efflux from V79 cells by verapamil.
  • Cells were incubated in MEBSS contain ⁇ ing 99m Tc-MIBI for 15 minutes (plateau loading) , then transferred to 99m Tc-MIBI-free MEBSS washout buffer for various times in the absence (O) or presence (•) or verapamil (lO ⁇ m) .
  • Cell associated tracer activity was determined and expressed as fmol cellular Tc-MIBI/mg protein per nmolar Tc-MIBI concentration in the uptake buffer. Each point represents the mean of 4 deter ⁇ minations. Error bars represent + SEM when larger than symbol. Note semilog plot.
  • FIG. 4 Effect of guinidine on accumulation of Tc- MIBI in V79 cells.
  • Cells were incubated for various times in MEBSS containing 99m Tc-MIBI in the absence (O) presence (•) of quinidine (10 uM) and then cell associated activity determined. Points represent the mean + SEM of three determinants each.
  • FIG. 5 Cell survival studies and LD 50 determination showing cytotoxicity of a 99 Tc-isonitri!e complex that is transported by the P-glycoprotein in vitro . Survival of parental V79 and multidrug resistant LZ cells (A) and parental Alex and multidrug resistant Alex/A.5 cells (B) in increasing concentrations of carrier-added 99 Tc-MIBI. Methods: Cell survival assays were performed by plating 4,000-20,000 cells per well in 96 well microtiter plates in triplicate in three separate experiments. V79 and LZ cells were grown essentially as described in Example 2 while Alex and Alex/A.5 were grown as described by Goldstein, L., et al . , J. Natl . Cancer Inst. 81:116 (1989).
  • Multidrug resistant cells were cultured in drug free media for 72-96 hours prior to culture in 99 Tc-MIBI.
  • Surviving cells were assayed by staining with sulforhodamine B (Mazzanti, R. , et al . , J. Cell Pharmacol . 1:50 (1990)) at 72 hours for V79 and LZ cells or 96 hours for Alex and Alex X.5 cells. Survival was expressed as the percentage of surviving cells relative to growth in alpha-MEM media without 99 Tc-MIBI. Standard error bars are displayed. LD 50 determinations were obtained by interpolation from the cell survival curves.
  • FIG. 6 Direct examination of the interaction of 99 Tc-MIBI with MDR P-glycoprotein using the photoaffinity probe 125 I-iodoaryl-azidoprazosin (IAP) showing inhibition by 99 Tc-MIBI of IAP binding to P- glycoprotein.
  • IAP I-iodoaryl-azidoprazosin
  • FIG. 8 Net accumulation of a novel Tc-isonitrile complex, Tc-TMPI (trimethoxyphenyl isonitrile) (•) , compared to the P-glycoprotein transport substrate Tc-MIBI ( ⁇ ) in drug resistant 77A cells, a mammalian cell line expressing intermediately high levels of P- glycoprotein.
  • Tc-TMPI trimethoxyphenyl isonitrile
  • ⁇ P-glycoprotein transport substrate
  • ⁇ P-glycoprotein transport substrate
  • Cells were plated in 100-mm petri dishes containing seven 25-mm glass coverslips on the bottom and grown to confluence in alpha-MEM medium (GIBCO) supplemented with L-glutamire (1%) , penicillin/streptomycin (1%) and fetal calf serum (10%) in the presence of 0.1 ug/ml ⁇ -iamycin.
  • Tracer accumulation was determined at the times indicated in cells incubated in modified Earle's balanced salt solution containing 145mM Na + , 5.4mM K + , 1.2mM Ca 2+ , 0.8mM Mg 2+ , 152mM Cl " , 0.8mM H 2 P0 4 -, 0.8mM S0 4 2 ⁇ , 5.6mM dextrose, 4.0mM HEPES (pH 7.4; 37°C) , 1% bovine calf serum, and 0.1-0.6 nM 99m Tc-isonitrile complex (25-100 uCi/ml; 0.1-0.4 Ci/nmole) .
  • Net accumulation of the 99ra Tc-isonitrile complexes in each preparation was normalized to cell protein determined by the method of Lowry and to extracellular tracer concentration determined from an aliquot of the load solution using a well-type gamma counter (Piwnica- Worms et al . , Circ . 82:1826 (1990)). Each point is the mean of 3-4 determinations; SEM did not exceed 15% t of mean values.
  • FIG. 9 Net accumulation at 60 minutes of the hexakis complex Tc-TMPI in cultured heart and 77A cells. Methods are the same as described in Figure 8. Piwnica-Worms et al . , (Circ. 82 : 1826 (1990)) also describes methods for obtaining primary cultured avian heart cells.
  • FIG. 10 Scintigraphic images of the novel hexakis complex Tc-TMPI binding in vivo in adult rabbits. Animals were anesthetized with xylezine (10 mg/kg) and ketamine (40 mg/kg) IV and positioned over a gamma camera (GE Starcam; LEAP collimator) . A bolus of 99m Tc-TMPI (1.5 mCi) was then injected via an ear vein. Planar images were collected for 60 sec/frame up to 1 hour. Energy discrimination was provided by 20% windows centered over the 140 KeV photopeak of 99m Tc. Each image was corrected on-line for camera nonuniformity with a 300 million count flood and stored at a digital resolution of 64 x 64. No attenuation or scatter correction was used. Time- activity curves were generated by placing a region-of- interest over each organ and expressing activity in CPM/pixel.
  • the present invention is based on the discovery that the gamma emitter, 99m Tc-MIBI, commonly used as an imaging agent for myocardial perfusion analysis, is transported out of cells by the multidrug resistance transport system.
  • This agent normally taken up by living cells, is actively excluded from living cells by the multidrug resistance system.
  • the net accumulation of this agent is reduced compared to the levels accumulated by normal cells or cells to which have been co-administered t e agent plus an agent that reverses the multidrug resistance phenotype (reversing agent) . In the latter two instances, the net accumulation of the agent is higher and can be distinguished from that of multidrug-resistant cells.
  • the invention is directed to methods of detecting the multidrug resistance phenotype in an animal, tissues, or cells by administering to the animal, tissues, or cells, an agent which is trans ⁇ ported by the multidrug resistance transport system and which is detectable in living cells, at distances removed, by the presence of intervening tissue, from the in situ location of the cells.
  • the methods encompass measuring the intracellular accumulation of the agent in the animal, tissues, or cells, and comparing the measurement with the measurement obtained with a control that does not express the multidrug resistance phenotype.
  • the multidrug resistance phenotype is detected in an animal, tissues, or cells by administering to the animal, tissues, or cells, an imaging agent, particularly a scintigraphic imaging agent, which is transported by the multidrug resistance transport system.
  • an imaging agent particularly a scintigraphic imaging agent, which is transported by the multidrug resistance transport system.
  • the methods then encompass imaging the animal, tissues, or cells, and comparing the image with the image obtained with a control that does not express the multidrug resistance phenotype.
  • the invention embodies methods to assay the multidrug resistance phenotype in instances that previously required invasive in vivo surgical procedures or time-consuming in vitro outgrowth procedures.
  • the procedure involves trauma, often to patients already traumatized by prior treatment.
  • the phenotype of explanted cells subject to the different selective pressures of tissue culture, and no longer subject to the in vivo selective pressures in the patient, would be subject to genotypic and phenotypic alteration • that could confound diagnosis and treat ⁇ ment.
  • the present invention therefore, embodies methods of detecting the multidrug resistance pheno- type in tissues in vivo without the need for invasive procedures and in whole tissue in vitro.
  • the net cellular accumulation of the agent alone is compared with the net cellular accumulation of the agent when it is co-administered with an agent that reverses the multidrug resistance phenotype ("revers ⁇ ing agent”) .
  • revers ⁇ ing agent an agent that reverses the multidrug resistance phenotype
  • the agent In the presence of a reversing agent or other inhibitor, the agent is not excluded from cells, whereas in the absence of a reversing agent, relative exclusion of the agent occurs.
  • the agent will be detected to a greater extent when administered with a reversing agent than the extent to which it is detected when administered alone. The agent is, therefore, useful as a marker for detecting the multidrug resistance phenotype.
  • the agent is an imaging agent.
  • the preferred imaging agent of the present invention is Tc-MIBI.
  • the presence or absence of expression of the multidrug resistance phenotype is evaluated by scintigraphic imaging (either planar or SPECT) with Tc-MIBI before and after the administration of the reversing agent.
  • the tissue significantly expressing the multidrug resistance phenotype shows little Tc-MIBI localization in the absence of the reversing agent but enhanced uptake of Tc-MIBI during infusion of the reversing agent.
  • the tissue not expressing the multidrug resistance pheno- type t lows Tc-MIBI localization initially, but does not demonstrate reversing agent-induced enhancement of net uptake.
  • the imaging methods of the present invention encompass any non-toxic imaging agent that is transported by the multidrug resistance transport system.
  • Alternative preferred imaging agents include, but are not limited to, other hexakis (R-isonitrile) technetium (I) complexes.
  • Other embodiments encompass analogous complexes of paramagnetic and susceptibility metals such as lipophilic cation complexes of Mn, Fe, Gd, Dys for use in magnetic resonance imaging in vivo and in vitro and labeled positron-emitting ligands useful in positron emission tomography ("PET" scan).
  • Alternative embodiments encompass other gamma-emitting labels such as rhenium, indium, iodine, and copper.
  • One preferred embodiment relates to the source of the Tc isotope.
  • the specific activity of the 99 Tc- MIBI complex synthesized from 99 Tc0 4 ⁇ obtained directly from commercial molybdenum/technetium generators, is extremely high.
  • 99m Tc-MIBI was generally synthesized at 1-6 X 10 8 Ci/mole.
  • [ 3 H] TPP + another lipophilic cation, is commonly supplied commercially at 5-100 Ci/mole. This provides an opportunity to decrease the molar concentration of cation accumulation by the biological preparation, yet remain within detectable limits.
  • Typical reversing agents include verapamil and quinidine.
  • the invention can be practiced with any agent that reverses the multidrug resistance phenotype.
  • alternative reversing agents include, but are not limited to vinblastine, vincristine, adriamycin, colchicine, daunomycin, dactinomycin, vanadate, cyclosporine and tetraphenyl- borate.
  • a patient receives the detection agent in both the presence and absence of a reversing agent. The treatment is in either order. If the two drugs are first administered together, then following the detection process, the reversing agent is given sufficient time to leave the system before the administration of the agent alone.
  • Multidrug resistance tissue is detected in the pres ⁇ ence of a reversing agent but not in its absence. Using this method, multidrug resistant tissue is located without invasive procedures.
  • One of the most daunting obstacles to success ⁇ ful chemotherapeutic treatment of tumors is the acquisition by tumors of the multidrug resistance phenotype.
  • the acquisition of multidrug resistance is usually discovered when the patient no longer responds to the prescribed chemotherapeutic regimen.
  • multidrug-resistant tumors are detected in cancer patients without the need for surgery by administering to cancer patients an imaging agent of the present invention in the presence and absence of a reversing agent and comparing the images.
  • agents are not detected by imaging but by quantitative measurement of the intracellular accumulation (e.g., in a gamma counter or as by scanning radiograms by densitometry) .
  • the imaging agent is 99m Tc- MIBI.
  • the presence or absence of expression of the multidrug resistance phenotype in tumors is evaluated non-invasively by the scintigraphic imaging with 99 Tc- MIBI of cancer patients before and after the administration of a reversing agent.
  • Those tumors significantly expressing the multidrug resistance phenotype will show little 99 Tc-MIBI localization in the absence of the reversing agent but enhanced uptake of 99m Tc-MIBI within the tumor or metastasis during infusion of the reversing agent.
  • Those tumors not expressing the multidrug resistance P-glycoprotein should show 99m Tc-MIBI localization initially, but should not demonstrate reversing agent-induced enhancement of net uptake.
  • 99m Tc-MIBI is a preferred imaging agent for whole tissue imaging.
  • Imaging agents include, but are not limited to the agents mentioned above.
  • a highly preferred combination is 99m Tc-MIBI as the imaging agent and verapamil, quinidine, or cyclosporine as the reversing agent.
  • the invention can be practiced with any agent that reverses the multidrug resistance phenotype. Examples of alternative reversing agents are discussed above.
  • the methods of the present invention are also applicable to whole tissue and cells in vitro .
  • the invention is advantageous over current methods of determining the multidrug resistance phenotype in vitro because it is rapid and simple. Using presently available methods, before the multidrug resistance phenotype can be evaluated in whole tissue, a single cell suspension must be created (e.g., for flow cytometry) or even more laborious techniques must be used, such as monolayer cell culture. Using the method of the current invention, it is possible to detect the multidrug resistance phenotype in tissue without, or with minimum, disaggregation. Thus, therapeutic regimens may be decided with less delay than with presently available methods.
  • the overexpression of the multidrug resistance gene in a tumor occurs as a result of the selection and multi- plication of single or a few mutant cells as the tumor is subjected to a chemotherapeutic drug.
  • the tumor is excised and grown in tissue culture, the genotype may change because the selection pressure is not the same. This may interfere with the proper analysis of the tumor and hence with prescription of a effective therapeutic regimen.
  • the tumor could be analyzed without dispersion and growth in culture. Relevant prescription would then be more likely.
  • tumors are usually genotypically and phenotypically heterogeneous. New genotypes may arise in a very small or minute portions of a tumor and may not be detectable by routine methods. For example, the multidrug resistance phenotype occurring in a small area of a tumor, may be missed if the tumor cells are dispersed or merely biopsied. With the methods of the present invention, since a small area would be intact, imaging the tumor would reveal such small pockets of multidrug resistant cells.
  • the invention embodies methods of assaying the multidrug resistance phenotype in whole tissue or tissue biopsies by incubating the tissue or biopsy with the agents of the present invention.
  • the tissue is exposed to the agent in the presence and absence of a reversing agent, such as those mentioned above. Accumulation of the agent in the tissue is measured in both cases and the measurements are compared.
  • the agent is administered alone and the measurement obtained is compared with the measurement obtained with normal control tissue.
  • the agent is an imaging agent. 99m Tc-MIBI is a preferred imaging agent for whole tissue imaging.
  • Alternative imaging agents include, but are not limited to the agents mentioned above.
  • a highly preferred combination is 99m Tc-MIBI as the imaging agentandverapamil or quinidine as the reversing agent.
  • the invention also embodies methods of designing chemotherapy regimens by assaying the multidrug resistance phenotype in patients or their explanted tissue either prior to or during treatment. During 5 the course of chemotherapy, when it is determined that a multidrug resistance-negative tumor (previously showing agent localization) converts or recurs with multidrug resistance (expressed as loss of agent localization) , this valuable information is used to
  • patients are evaluated for the multidrug resistance phenotype prior to initiation or continuation of chemotherapy. Those patients deemed phenotypically multidrug
  • chemotherapeutic drugs 20 alternative chemotherapeutic drugs.
  • the ability of a drug to act as a chemosensitizer (reversing agent used in chemotherapy to facilitate the uptake of a chemotherapeutic drug in drug-resistant tumor cells) is determined.
  • the chemosensitizer is then used to facili ⁇ t ate the administration of or to test the efficacy of anti-tumor drugs.
  • the location of tumors not detectable by standard means is determinable if
  • the methods of the present invention provide means to monitor progression or regression of the disease during chemotherapy.
  • the discovery of the present invention also provides embodiments in which agents are transported by other members of the ATP binding cassette transport family of proteins.
  • one embodiment of the invention is a test for sensitivity to new anti- malarial agents in drug-resistant malaria parasites in which the pfMDR gene is over-expressed or a test of bacterial transport function where the transporter belongs to the family.
  • the invention may also be extended to imaging those tissues that naturally express elevated levels of the multidrug-resistance transport protein relative to other normal tissues. This includes liver, bone marrow, adrenals, kidney, and lung. These tissues would appears as photodeficient areas.
  • the present invention relates to design and therapeutic uses of similar complexes that bind to the multidrug resistance transporter protein.
  • the invention thus provides a new class of reversing agents and direct chemotherapeutic compounds.
  • the present invention is therefore generally directed to a method for directly killing a cell or reversing the multidrug resistance phenotype in a multidrug resistant cell comprising administering the complexes of the present invention to the cell.
  • the invention is directed to a method of enhancing the intracellular accumulation of a drug in multidrug resistant cells wherein the accumulation is dependent upon transport by the multidrug resistant transport system which involves P-glycoprotein.
  • the compound of the present invention is co-administered with the drug.
  • the administration may be in vitro or in vivo.
  • the enhancement of accumulation of the drug is in multidrug resistant cells in vivo.
  • the complexes are administered to cancer patients in vivo as a means of treating tumors which have become multidrug resistant in the course of therapy.
  • chemotherapeutic agents are administered with the compounds of the present invention.
  • the coadministration is designed to enhance accumulation of the agent following reversal of the multidrug resistance phenotype by interaction of the compounds of the present invention with the multidrug transport system.
  • the coadministration is designed to cause the chemotherapeutic agent to accumulate in amounts effective for cytotoxicity whereas when the agent is administered alone, accumulation in effective amounts does not occur.
  • This coadministration regimen can be applied to any cell which exhibits the multidrug resistant phenotype as a result of overexpression of the multidrug resistance protein, e.g., P-glycoprotein.
  • the compounds of the present invention also provide methods to study cytotoxicity in vitro in a search for new cytotoxic compounds.
  • multidrug resistant cells may be exposed in vitro to the potential compound in the presence of the compounds of the present invention.
  • This regimen also allows determination of effective combinations for chemotherapy by demonstrating which chemotherapeutic drugs can be effectively accumulated in multidrug resistant cells as a result of the addition of the compounds of the present invention.
  • These regimens may be used in tissue biopsies to assess effective cytotoxic agents for a particular patient. Accordingly, the compounds of the present invention may be used to tailor chemotherapy to the individual patient by assessing the effect in biopsies of combinations of the compounds of the present invention and various known or potential chemotherapeutic agents.
  • the present invention is directed to a method of enhancing the effect of a reversing agent.
  • the compounds of the present invention are added to a regimen which already includes the use of a reversing agent being co-administered with a chemotherapeutic agent or other agent whose intracellular accumulation in multidrug resistant cells is desired.
  • the compounds of the present invention would be administered concurrently with another known reversal agent to enhance the cytotoxicity or reversing properties of the second agent.
  • the invention is directed to the use of the compounds of the present invention alone as a method of killing multidrug resistant cells in vivo and in vitro.
  • This encompasses direct use of radioactive isotopes incorporated into the metal core of the metal isonitrile complexes for local deposition of ionizing radiation into the multidrug resistant cells.
  • the multidrug resistant cells are found in tumors in patients in vivo.
  • Radioactive ligands could also be synthesized and bound to the core metal for use as selective radioactive agents for such radiotherapy.
  • radio frequency energy applied at the nuclear magnetic residence frequency of the metal core of the agent could be used therapeutically to selectively deposit thermal energy in multidrug resistant cells, particularly tumors, while patients are inside a nuclear magnetic residence imaging device.
  • the method would be directed to the use of the compounds of the present invention as enhancing agents to increase tissue accumulation of 99 Tc-MIBI or other technetium isonitrile complexes during diagnostic imaging of tissue perfusion or tumors using gamma camera scintigraphy.
  • the complexes of the present invention would serve as the reversing agent relevant to the diagnostic use of the isonitrile complexes discussed in the section above headed "Diagnostic Imaging.”
  • the complexes of the present invention may function as novel antimalarial drugs. This is because the transport system which transports drugs in protozoans involves an ATP-binding transport system similar to the P-glycoprotein multidrug resistant transport system.
  • the complexes of the present invention could also be used as antibiotics by inhibiting bacterial membrane transport systems, which systems operate through ATP-depending binding proteins homologous to the P-glycoprotein mult irug resistant transport system.
  • the compounds and methods of the present invention could be applied to any of the transport systems which function through transport proteins with amino acid similarities and structural relationships with the multidrug resistant transport protein, P-glycoprotein. Many of these proteins have been discussed in the background section above.
  • typical structures are hexakis isonitrile complexes of a core metal which may be, but is not limited to, rhenium or technetium.
  • a core metal which may be, but is not limited to, rhenium or technetium.
  • Other multi-ligand isonitrile complexes of iron, cobalt, manganese, nickel, chromium and copper can be synthesized.
  • all metals but technetium have stable (non-radioactive) isotopes found in nature and which are suitable for drug manufacture.
  • radioactive isotopes of all the metals may be synthesized for use during drug design, for example, for performing biodistributions or for therapeutic exploitation of the radioactive emissions into cancer tissues.
  • the side groups of these compounds are substituted alkyl and aryl isonitrile ligands.
  • Preferred compounds of the present invention include but are not limited to hexakis (arylisonitrile
  • rhenium (rhenium) (I) complexes as drugs for the methods of the present invention.
  • Preferred embodiments include hexakis arylisonitrile rhenium I complexes where the arylisonitrile ligand is optimized for binding affinity to the multidrug resistant transport protein.
  • the ligand is trimethoxyphenylisonitrile (see Figure 7) .
  • ligands are analogous to the substituted aryl structure of verapamil, a known high affinity P- glycoprotein binding drug and reversing agent.
  • the aryl group in addition to the core metal, may be based on any of the other reversing compounds known to bind to P-glycoprotein. Structures may also be based on compounds which bind to vesicles produced from multidrug resistant cell membranes, particularly compounds which may interfere with the labeling of such vesicles or P-glycoprotein directly.
  • Preferred isonitrile ligands include, but are not limited to, 4-methoxyphenylisonitrile; 3-,4-dimethoxy- phenylisonitrile; 3-,5-dimethoxyphenylisonitrile; 3-,4-,5-trimethoxyphenylisonitrile; 4-methoxybenzyl- isonitrile; 3-,4-dimethoxybenzylisonitrile; 3-,4-,5- trimethoxybenzylisonitrile; 3-,4-dimethoxyphenethyl- isonitrile; 3-,4-,5-trimethoxyphenethylisonitrile; 4- methoxyphenethylisonitrile; phenylisocyanide isonitrile; phenylacetyl isonitrile; phenylacetamide isonitrile; or 3-,4-0-CH 2 -0-phenylisonitrile.
  • Each isonitrile ligand has been readily synthesized from the corresponding primary amine by standard reaction with chloroform and base in the presence of phase transfer catalyst (Nicolini, M. et al . , Eds., "Technetium and Rhenium” in Chemistry and Nuclear Medicine, Cortina Internet, Verona Italy (1986) .
  • the hexakis Tc or Re complex of the isonitrile ligand can be synthesized by addition of pertechnetate or perrhenate (Re0 4 -) and reducing agent (Na 2 S 2 0 4 ) to the free ligand as described (Piwnica-Worms et al . , Invest . Radiol . 24 : 25 (1989)).
  • Assays for the efficacy of such compounds include, but are not limited to, the ability to enhance the uptake of a chemotherapeutic agent such as daunorubicin or vincristine, the ability to block the efflux of chemotherapeutic agents from cells, the ability to interfere with photoaffinity labeling of multidrug resistant membrane vesicles, and the general ability to compete with chemosensitizing agents in any of the assays involving reversal of the multidrug phenotype or binding to P-glycoprotein.
  • ATP-binding cassette transport protein is intended, for the purpose of the present invention, a protein that is a member of the ATP- binding cassette superfamily of transport proteins.
  • This family is recognized by sequence identity over a "cassette" of about 200 amino acids.
  • This cassette is an ATP-binding domain which is the distinguishing feature of this family of transport proteins. These domains share about 30-40% sequence identity among the members of the superfamily.
  • a putative member of the superfamily would be recognized by sequence comparison of the primary structure with a primary consensus sequence or individual sequences of these proteins using routine computerized sequence scanning methods. The degree of identity in the conserved domain between any pair of these proteins is essentially the same whichever two proteins are compared. It is important to emphasize that these ATP-binding proteins are distinct from other proteins that bind ATP in that the sequence homology is much greater than is required for merely nucleotide binding. That is, the homology extends considerably beyond the two consensus nucleotide-binding sequences of Walker et al . (EMBO J. 1:945 (1982)) which comprise five and nine amino acids.
  • putative member of this distinctive family by computerized alignment of the primary sequence with the primary sequence of one or more of the known ATP-binding cassette transport proteins. Computerized sequence comparison and alignment is done with any one of the well-known and routinely used sequence homology/identity scanning programs. These programs wov'd be readily available to the skilled artisan. h, putative member of the family should contain, in addition to the two short consensus nucleotide-binding sequences of Walker et al . (EMBO J.
  • cells are intended any one of the components that make up an organized tissue, consist ⁇ ing of a nucleus which is surrounded by cytoplasm which contains the various organelles and is enclosed in the cell or plasma membrane.
  • cells are in vivo as part of the living organism, in explanted tissue taken from a living organism, or in cell culture.
  • multidrug resistance for the purpose of the present invention is intended the phenotype that occurs in a cell as the result of the overexpression of the gene product of the multidrug resistance gene or its homolog.
  • multidrug resistance gene is intended that DNA sequence which encodes P-glycoprotein and its functional equivalents and whose amplification confers upon a cell cross- resistance to toxic drugs.
  • homolog is intended the DNA sequence in another species, which sequence corresponds to the multidrug resistance gene.
  • administer is intended any method for introducing the compositions of the present invention into a subject.
  • Typical methods include, but are not limited to, oral, intranasal, parenteral (intravenous, intramuscular, or subcutaneous) , or rectal.
  • parenteral intravenous, intramuscular, or subcutaneous
  • rectal or rectal.
  • the term “administer” also relates to the application of substance ex vivo as in cell or organ culture.
  • administration When administration is for the purpose of treatment, administration may be for either prophylactic or therapeutic purposes.
  • the substance When provided prophylactically, the substance is provided in advance of any symptom.
  • the prophylactic administration of the substance serves to prevent or attenuate any subsequent symptom.
  • the substance When provided therapeutically, the substance is provided at (or shortly after) the onset of a symptom.
  • the therapeutic administration of the substance serves to attenuate any actual symptom.
  • administer is intended that each of at least two compounds be administered during a 5 time frame wherein the respective periods of biological activity overlap. Thus the term includes sequential as well as coextensive administration of the compounds of the present invention.
  • compound should be read to include synthetic compounds, natural products and macromolecular entities such as polypeptides, poly- nucleotides, or lipids and also small entities such as
  • the term “compound” is meant to refer to that compound whether it is in a crude mixture or purified and isolated.
  • 25 present inv* ntion is intended the ATP-binding protein- dependent movement of a compound across the membrane of a living cell and especially where the protein is P-glycoprotein or its homologs.
  • Transport may encompass mechanisms wherein a substrate is bound
  • imaging for the purpose of the invention is intended the production in clarity, contrast, and detail in images, either by analogue or digital devices, especially in radiological images.
  • scintigraphy is intended the production of two dimensional or three dimensional reconstructed images of the distribution of radio ⁇ activity in tissues after the internal administration of radionuclide, the images being obtained by a scintillation camera.
  • chemosensitizer or "reversing agent” are intended for the purpose of the present invention, a compound that allows the net accumulation of toxic compounds in multidrug-resistant cells equivalent to the net accumulation of said toxic compounds in non-multidrug-resistant cells.
  • the presence of these agents may also merely increase the amount of the toxic compound able to accumulate in a multidrug resistance cell compared to the amount accumulated in the absence of the agent.
  • tumor is intended for the purpose of the present invention, a new growth of tissue in which the multiplication of cells is uncontrolled and progressive.
  • the tumor that is particularly relevant to the invention is the malignant tumor, one in which the primary tumor has the properties of invasion and metastasis and which shows a greater degree of anaplasia than do benign tumors.
  • R is intended alkyl, substituted alkyl, aryl, or substituted aryl groups. R groups are found in the general formula -CR 3 where R can be identical or different and includes the elements H, C, N, O, S, F, Cl, Br, and I.
  • Representative examples include, but are not limited to, -CH 3 , -CH 2 CH 3 , CH(CH 3 ) 2 , -C(CH 3 ) 3 , -C(CH 3 ) 2 OCH 3 , -C(CH 3 ) 2 C00CH 3 , -C(CH3) 2 OCOCH 3 -C(CH 3 )C0NH 2 , -C 6 H 5 , -CH 2 (C 6 H 4 )0H, or any of their iso eric forms having the general composition as the isonitrile radionuclide complexes in U.S. Patent No. 4,452,774 which is incorporated herein by refer ⁇ ence.
  • alkyl is intended any straight, branched, saturated, unsatura ed or cyclic C ⁇ Q alkyl group.
  • Typical C j -C ⁇ alk * 1 groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n- butyl, t-butyl, i-butyl, pentyl and hexyl groups.
  • Preferred alkyl groups may include isopropyl, isobutyl, 2-ethyl-n-propyl, and t-butyl.
  • aryl is intended any cyclic hydrocarbon based on a six-membered ring.
  • Typical aryl groups include, but are not limited to, phenyl, naphthyl, benzyl, phenethyl, phenanthryl, and anthracyl groups.
  • Preferred groups include, but are not limited to, methoxyphenyl; dimethoxyphenyl; trimethoxyphenyl; methyoxybenzyl; dimethoxybenzyl; trimethoxybenzyl; dimethoxyphenethyl; trimethoxyphenethyl; methoxyphenethyl; phenylisocyanide; phenylacetyl; phenylacetamide; or 0-CH 2 -0-phenyl.
  • substituted alkyl and “substituted aryl” is intended any alkyl or aryl group in which at least one carbon atom is covalently bonded to any functional group comprising the atoms H, C, N, O, S, F, Cl, Br and I.
  • Typical substituted alkyl groups include but are not limited to amino, c ⁇ - & alkylamino, c 2 - i2 dialkylamino, C 1 _ 6 alkoxy, and C 2 _ 6 alkylcarboxy.
  • Typical substituted aryl groups include, but are not limited to the above-listed aryl groups substituted by halo, hydroxy, ⁇ -C 8 alkoxy, amino, and the like.
  • animal is intended any living creature that contains cells in which the intra- membrane potential is reduced by the administration of agents of this invention. Foremost among such animals are humans; however, the invention is not intended to be so-limiting, it being within the contemplation of the present invention to apply the compositions of the invention to any and all animals which may experience the benefits of the application.
  • over-express is intended for the purpose of the present invention, the production of an ATP-binding cassette transport protein in a cell type in amounts exceeding that normally produced in that cell type.
  • expression may vary amongst normal cell types, within each type an ATP-binding cassette transport protein is expressed within a normal physiological range. Over-expression may be due to gene amplification, an increase in RNA transcription rates, increase in RNA stability, increase in mRNA translation, or any other molecular process which results in amounts of ATP-binding cassette transport protein exceeding those amounts found in normal cells.
  • the normal range of expression in a given cell type can be determined by routine methods as by assaying the ATP-binding cassette transport protein, its mRNA, or its gene.
  • Assays may be those commonly used in the art such as immunoassay, PAGE, western blot, Southern and northern blots, Cot analysis, Rot analysis, and competition hybridization procedures.
  • reversing the multidrug resistance phenotype is intended causing cells, which over express the multidrug resistance gene product and therefore survive in the presence of cytotoxic agents, to become sensitive to the agents.
  • the agents may be chemotherapeutic agents or agents which are otherwise toxic to cells.
  • the agents are transported by the multidrug transport protein.
  • the reversal may occur by irreversibly binding to the transport protein thereby preventing the efflux of the therapeutic compound irreversibly or by competitively inhibiting said therapeutic compound by binding the sites in the transport system ordinarily occupied by that compound.
  • the reversal may be transient or permanent.
  • efflux is intended the transport of a chemotherapeutic compound or other compound whose intracellular accumulation is desired, out of a cell.
  • (R-isonitrile) metal complex compounds comprising a core metal atom, either radioactive or non-radioactive, coordinate bonded to the terminal carbon of the R-isonitrile ligands described above.
  • Control buffer was a modified Earle's balanced salt solution (MEBSS) with the following composition (nM) : Na + , 145; K + , 5.4; Ca 2 , 1.2; Mg 2+ , 0.8; Cl, 152; H 2 P0 4 , 0.8; S0 4 2 ⁇ , 0.8; dextrose, 5.6; HEPES, 4.0; and bovine calf serum, 1% (v/v); pH 7.4 ⁇ 0.05; 37°C. Verapamil and quinidine were dissolved into DMSO prior to addition to buffer. DMSO alone has no significant effect on contractile activity, action potential configuration (Lieberman, M. , et al . , Dev. Biol . 32:380-403 (1973)) or Tc-MIBI uptake kinetics (Piwnica-Worms, D. , et al . , Circ. 82:1826-1838 (1990)).
  • MEBSS modified Earle's balanced
  • the kit reaction vial contains the isonitrile ligand in the form of tetrakis (2-methoxy isobutyl isonitrile) Copper (I) tetrafluoroborate (1.0 mg) , a stannous chloride reducing agent (0.075 mg L-cysteine hydrochloride (1.0 mg) , Sodium Citrate (2.6 mg) and mannitol (20 mg) .
  • the intrinsically radiolabeled complex was formed by adding [ 99M Tc]Tc ⁇ 4 " (20-30 mCi, 2-25 pmol/mCi) in 1-2 ml saline (0.15M, NaCl) , obtained from a commercial molybdenum/technetium generator (DuPont Medical Products, Billerica, MA) , to the kit reaction vial, heating at 100°C for 15 min, and allowing to cool to room temperature producing an almost quantitative yield of the [ 99m Tc] (MIBI) 6 + complex.
  • LZ, 77A, and V79 cells were grown to confluence in approximately four days on 25 mm glass cover slips placed on the bottom of 100 mm plastic culture dishes in Minimal Essential Media (MEM) Alpha Medium supple ⁇ mented with 10% fetal calf serum, 1% L-glutamine, and 1% penicillin/streptomycin solution at 37°C in a 5% C0 2 /95% air humidified environment. Serial passage was performed by gently shaking the cells off the culture dish during exposure to 0.25% trypsin solution for 2-3 minutes (room temperature) and diluting the cell suspension in growth medium 1:4 for LZ cells and 1:20-40 for 77A and V79 cells. For 77A and LZ cells, growth medium was also supplemented with 0.1 and 4 ⁇ g/ml adriamycin, respectively.
  • Preparations were removed at various times and rinsed three times in separate 25 ml volumes of ice-cold (2 ⁇ C) isotope-free buffer for 8 seconds each to clear extracellular spaces.
  • preparations were incubated in loading solution for 15 minutes, rinsed as above in ice-cold buffer, and then immersed in 30 ml of isotope-free MEBSS (37°C) for the times indicated. Cell-associated activity was then determined.
  • Tc-MIBI Net accumulation of Tc-MIBI in LZ cells, a cell line highly selected for multidrug resistance pheno ⁇ type, is only 0.3 fmoles Tc-MIBI/mg protein per nmolar extracellular Tc-MIBI ( Figure 1) . This value is lower than that expected by simple equilibration of the agent into the cytosolic water space (approx. 5 fmoles/ g protein per nM 0 ) and implies active extru ⁇ sion of Tc-MIBI by the multidrug resistance P-glyco ⁇ protein.
  • High inhibitory doses of verapamil (ImM) increases net accumulation of Tc-MIBI greater than 100-fold over net uptake in the absence of the inhibitor.
  • V79 cells a cell line which modestly expresses multidrug resistance P-glycoprotein, demon ⁇ strates control accumulation of Tc-MIBI to plateau levels 20-fold higher than LZ cells ( Figure 1) . This is consistent with expectations for a cellular pheno ⁇ type with a less robust efflux pathway for Tc-MIBI.
  • Verapamil a known inhibitor of multidrug resist ⁇ ance P-glycoprotein (Gottesman, M.M. et al . , J. Biol . Chem. 263 : 12163 (1990)), increases net accumulation of Tc-MIBI in LZ cells to the higher levels expected for potential dependent uptake of the agent (Figure 2) . Verapamil also enhances the net accumulation of Tc- MIBI in V79 cells causing an 6-fold increase in net Tc-MIBI accumulation ( Figure 2) .
  • Verapamil (10 uM) is shown to directly inhibit unidirectional efflux of Tc-MIBI from V79 cells ( Figure 3) .
  • Micromolar concentrations of carrier-added 99 Tc- MIBI is shown to be cytotoxic in hamster lung fibroblast cells (A) and human carcinoma cells (B) ( Figure 5).
  • Tc-MIBI which is transported out of cells by P-glycoprotein, shows greater cytotoxic activity in drug-sensitive V79 and Alex cells compared to their multidrug-resistant derivative (LZ and Alex/A.5, respectively).
  • 99 Tc-MIBI was 11-fold and 13-fold more toxic in drug-sensitive V79 and human Alexander cells compared to their multidrug-resistant derivatives, respectively.
  • Tc-MIBI Inhibition by Tc-MIBI of photoaffinity labeling of P-glycoprotein with iodoaryl-azidoprazosin ( Figure 6) .
  • Tc-MIBI half-maximally inhibited photolabeling of P-glycoprotein at between 100- and 1,000-fold molar excess (lanes 3-6) , comparable to values for the known reversal agent verapamil (lanes 7-8) .
  • Tc-TMPI In drug resistant 77A cells, a mammalian cell line expressing intermediately high levels of multidrug resistance P-glycoprotein, the net accumulation of the novel complex Tc-TMPI is compared with the net accumulation of Tc-MIBI ( Figure 8) . Note that steady-state levels of Tc-TMPI content are 100- fold higher than Tc-MIBI. This is consistent with binding of Tc-TMPI to P-glycoprotein in these P-glycoprotein-enriched cells. Tc-MIBI has been previously shown to be an efflux transport substrate recognized by P-glycoprotein thereby resulting in low net accumulation. (Figure 1) .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hematology (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biochemistry (AREA)
  • Optics & Photonics (AREA)
  • Physiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des procédés de détection du phénotype de résistance multimédicamenteuse in vivo et in vitro. L'invention concerne en particulier des procédés de diagnostic du phénotype de résistance aux multimédicaments par imagerie, en particulier imagerie scintigraphique, dans des tumeurs solides in vivo ou dans des tumeurs et des biopsies in vitro. Les procédés de la présente invention permettent le diagnostic de la tumeur résistant aux multiples médicaments et autres phénotypes résistant aux multimédicaments sans avoir recours à des méthodes chirurgicales de type à invasion. La présente invention concerne également des procédés de traitement de tumeurs résistant à de multiples médicaments à l'aide de nouveaux agents qui se lient à la P-glycoprotéine. Les nouveaux composés de la présente invention sont administrés en association avec un agent chimiothérapeutique de manière à améliorer l'accumulation du médicament.
PCT/US1992/005329 1991-06-26 1992-06-26 Evaluation et traitement du phenotype de resistance multimedicamenteuse WO1993000064A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US07/719,714 US5407653A (en) 1991-06-26 1991-06-26 Evaluation of the multidrug resistance phenotype
US719,714 1991-06-26

Publications (2)

Publication Number Publication Date
WO1993000064A2 true WO1993000064A2 (fr) 1993-01-07
WO1993000064A3 WO1993000064A3 (fr) 1993-02-04

Family

ID=24891073

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/005329 WO1993000064A2 (fr) 1991-06-26 1992-06-26 Evaluation et traitement du phenotype de resistance multimedicamenteuse

Country Status (3)

Country Link
US (2) US5407653A (fr)
AU (1) AU2304192A (fr)
WO (1) WO1993000064A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996033285A1 (fr) * 1995-04-21 1996-10-24 Microcide Pharmaceuticals, Inc. Inhibiteurs de pompe a ecoulement
US5767113A (en) * 1995-05-10 1998-06-16 The Salk Institute For Biological Studies Compounds useful for concurrently activating glucocorticoid-induced response and reducing multidrug resistance

Families Citing this family (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5420321A (en) * 1993-08-03 1995-05-30 The Du Pont Merck Pharmaceutical Company Tris(isonitrile)copper(i) sulfates for preparing radionuclide complexes
WO1996000085A1 (fr) * 1994-06-27 1996-01-04 Mallinckrodt Medical, Inc. Procede d'imagerie par difference permettant d'identifier des cellules tumorales resistant a une therapie polymedicamenteuse
US6277655B1 (en) 1994-10-13 2001-08-21 Solvo Biotechnology Assay and reagent kit for evaluation of multi-drug resistance in cells
HU217108B (hu) * 1994-08-31 1999-11-29 SOLVO Biotechnológiai Kft. Eljárás daganatok multidrogrezisztenciáját okozó fehérje aktivitásának in vitro mennyiségi kimutatására biológiai mintákban
US6497881B1 (en) * 1995-11-30 2002-12-24 New York University High efficiency tissue specific compound delivery system using streptavidin-protein a fusion protein
US20040023375A1 (en) * 2002-07-30 2004-02-05 Precision Therapeutics, Inc. Method for preparing cell cultures from biological specimens for chemotherapeutic and other assays
US20040072722A1 (en) * 2002-10-10 2004-04-15 Kornblith Paul L. Methods for assessing efficacy of chemotherapeutic agents
US5728541A (en) * 1996-07-12 1998-03-17 Precision Therapeutics, Inc. Method for preparing cell cultures from biologial specimens for chemotherapeutic and other assays
US6162616A (en) * 1997-04-16 2000-12-19 Millennium Pharmaceuticals, Inc. Multidrug resistance-associated polypeptide
WO1999067284A2 (fr) 1998-06-20 1999-12-29 Washington University Complexes peptidiques de permeation membranaire destines a l'imagerie medicale, au diagnostic et a la therapie pharmaceutique
US8038984B2 (en) * 1998-06-20 2011-10-18 Washington University Membrane-permeant peptide complexes for treatment of sepsis
US7306784B2 (en) 1998-06-20 2007-12-11 Washington University Membrane-permeant peptide complexes for medical imaging, diagnostics, and pharmaceutical therapy
US20060263382A1 (en) * 1998-06-20 2006-11-23 Richard Hotchkiss Membrane-permeant peptide complexes for treatment of sepsis
US6589503B1 (en) 1998-06-20 2003-07-08 Washington University Membrane-permeant peptide complexes for medical imaging, diagnostics, and pharmaceutical therapy
US6734192B1 (en) * 1999-08-23 2004-05-11 Mp-1 Inc. Treatment of viral infections
WO2001024763A2 (fr) 1999-10-01 2001-04-12 Immunogen, Inc. Compositions et methodes de traitement du cancer utilisant des immunoconjugues et des agents chimiotherapeutiques
US6258526B1 (en) * 2000-01-19 2001-07-10 M.D.R. Test Ltd Ex-vivo test kit for testing the effectiveness of reversers of multidrug resistance
WO2002076439A2 (fr) * 2001-03-23 2002-10-03 The Board Of Trustees Of The University Of Illinois Composes capables de moduler l'activite de transporteurs de medicaments multiples et leur utilisation therapeutique
US7115265B1 (en) 2001-05-14 2006-10-03 Duke University Four genetic tumor markers specific for human glioblastoma
WO2003032926A2 (fr) * 2001-10-19 2003-04-24 Memorial Sloan-Kettering Cancer Center Compositions et procedes pour inversion de la pharmaco-resistance
US7670604B2 (en) 2002-12-13 2010-03-02 Aurelium Biopharma, Inc. Vimentin directed diagnostics and therapeutics for multidrug resistant neoplastic disease
US7413851B2 (en) * 2002-12-13 2008-08-19 Aurelium Biopharma, Inc. Nucleophosmin directed diagnostics and therapeutics for multidrug resistant neoplastic disease
US7550256B2 (en) * 2002-12-13 2009-06-23 Aurelium Biopharma, Inc. Vimentin directed diagnostics and therapeutics for multidrug resistant neoplastic disease
ATE408838T1 (de) * 2003-01-03 2008-10-15 Aurelium Biopharma Inc Auf hsc70 gerichtete diagnostika und therapeutika für eine gegen mehrere arzneistoffe resistente tumorerkrankung
WO2004080819A2 (fr) * 2003-03-14 2004-09-23 Aurelium Biopharma Inc. Methode de diagnostic et de therapie ciblant la triose-phosphate isomerase, destinee aux maladies neoplasiques a multiresistance aux medicaments
US7294701B2 (en) * 2003-04-02 2007-11-13 Technion Research & Development Foundation Ltd. Antibody fragment capable of modulating multidrug resistance and compositions and kits and methods using same
US20090220432A1 (en) * 2005-08-31 2009-09-03 The Johns Hopkins University Imaging agents and methods of using same for detecting multidrug resistance in cancer
CA2620936A1 (fr) * 2005-09-01 2007-03-08 Precision Therapeutics, Inc. Tests de chimiosensibilite faisant appel a des cellules tumorales presentant des caracteristiques phenotypiques persistantes
DOP2006000277A (es) 2005-12-12 2007-08-31 Bayer Pharmaceuticals Corp Anticuerpos anti mn y métodos para su utilización
AR076284A1 (es) 2009-04-29 2011-06-01 Bayer Schering Pharma Ag Inmunoconjugados de antimesotelina y usos de los mismos
DE102010013114B4 (de) 2010-03-26 2012-02-16 Rüdiger Lawaczeck Prädiagnostisches Sicherheitssystem
US9579408B2 (en) 2011-02-11 2017-02-28 Washington University PET/SPECT agents for applications in biomedical imaging
US10282507B2 (en) 2015-11-24 2019-05-07 Oracle International Corporation Method and system for determining circuit failure rate

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4452774A (en) * 1982-04-30 1984-06-05 President And Fellows Of Harvard College Isonitrile radionuclide complexes for labelling and imaging agents
US4837306A (en) * 1985-02-25 1989-06-06 The Ontario Cancer Institute Method for selecting hybridomas producing antibodies specific to the P-glycoprotein cell suface antigen and a cDNA clone encoding the C-terminal portion of the antigen
US4894445A (en) * 1985-08-05 1990-01-16 E. I. Du Pont De Nemours And Company Metal-isonitrile adducts for preparing radionuclide complexes
US4872561A (en) * 1985-08-30 1989-10-10 Massachusetts Institute Of Technology Carboxy, carboalkoxy and carbamile substituted isonitrile radionuclide complexes
US5025020A (en) * 1989-09-28 1991-06-18 Cancer Biologics Of America, Inc. Use of tetrandrine and its derivatives to treat malaria
US5186923A (en) * 1990-10-10 1993-02-16 Brigham And Womens Hospital Enhancement of cellular accumulation of lipophilic cationic organometallic compounds by reduction of intramembrane potential

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996033285A1 (fr) * 1995-04-21 1996-10-24 Microcide Pharmaceuticals, Inc. Inhibiteurs de pompe a ecoulement
AU715654B2 (en) * 1995-04-21 2000-02-10 Essential Therapeutics, Inc. Efflux pump inhibitors
US5767113A (en) * 1995-05-10 1998-06-16 The Salk Institute For Biological Studies Compounds useful for concurrently activating glucocorticoid-induced response and reducing multidrug resistance

Also Published As

Publication number Publication date
AU2304192A (en) 1993-01-25
US5407653A (en) 1995-04-18
US5403574A (en) 1995-04-04
WO1993000064A3 (fr) 1993-02-04

Similar Documents

Publication Publication Date Title
US5403574A (en) Evaluation and treatment of the multidrug resistance phenotype
Piwnica-Worms et al. Functional imaging of multidrug-resistant P-glycoprotein with an organotechnetium complex
Reddy et al. Preclinical evaluation of 99mTc-EC20 for imaging folate receptor–positive tumors
AU2017268291B2 (en) PET-imaging immunomodulators
Weiss et al. Positron emission tomography imaging of tumors expressing the human chemokine receptor CXCR4 in mice with the use of 64 Cu-AMD3100
Xiong et al. Peptide-based imaging agents targeting phosphatidylserine for the detection of apoptosis
Kulterer et al. A microdosing study with 99mTc-PHC-102 for the SPECT/CT imaging of primary and metastatic lesions in renal cell carcinoma patients
Koch et al. Pharmacokinetics of EF5 [2-(2-nitro-1-H-imidazol-1-yl)-N-(2, 2, 3, 3, 3-pentafluoropropyl) acetamide] in human patients: implications for hypoxia measurements in vivo by 2-nitroimidazoles
US20070053834A1 (en) Radioligands for the TRP-M8 receptor and methods therewith
US5277897A (en) In vivo enhancement of cellular accumulation of lipophilic cationic organometallic compounds by reduction of intramembrane potential
Blankenberg et al. Imaging macrophages and the apoptosis of granulocytes in a rodent model of subacute and chronic abscesses with radiolabeled monocyte chemotactic peptide-1 and annexin V
Vaidyanathan et al. Improved xenograft targeting of tumor-specific anti-epidermal growth factor receptor variant III antibody labeled using N-succinimidyl 4-guanidinomethyl-3-iodobenzoate
Konrad et al. [99mTc] Tc-PentixaTec: development, extensive pre-clinical evaluation, and first human experience
Yang et al. Dose escalation biodistribution, positron emission tomography/computed tomography imaging and dosimetry of a highly specific radionuclide-labeled non-blocking nanobody
Ogawa et al. Astatine-211-labeled aza-vesamicol derivatives as sigma receptor ligands for targeted alpha therapy
Wang et al. Synthesis and evaluation of a radiolabeled bis-zinc (II)–cyclen complex as a potential probe for in vivo imaging of cell death
US20240024519A1 (en) Radiopharmaceutical compositions of copper for targeted molecular imaging
Cheng et al. PET imaging of CXCR4 expression using [18F] AlF-NOTA-QHY-04 for hematologic malignancy and solid tumors
Aoki et al. Development of radiolabeled bis (zinc (II)-dipicolylamine) complexes for cell death imaging
US10238758B2 (en) Radiolabeled alpha-melanocyte stimulating hormone hybrid peptide for melanoma targeting
US11795197B2 (en) Peptide compositions for immuno-oncology molecular imaging and targeted drug delivery
US6844425B1 (en) Combination of intercalating organometallic complexes and tumor seeking biomolecules for DNA cleavage and radiotherapy
Chen et al. Preparation and SPECT imaging of the novel Anxa 1-targeted probe 99m Tc-p-SCN-Bn-DTPA-GGGRDN-IF7
RU2720801C1 (ru) Способ радионуклидной диагностики рака молочной железы с гиперэкспрессией Her2/neu
WO2023015212A1 (fr) Compositions radiopharmaceutiques pour actinium à faible toxicité dans une thérapie ciblée par radionucléides

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU MC NL SE

AK Designated states

Kind code of ref document: A3

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU MC NL SE

COP Corrected version of pamphlet

Free format text: PAGES 1/10-10/10,DRAWINGS,REPLACED BY NEW PAGES 1/10-10/10;DUE TO LATE TRANSMITTAL BY THE RECEIVINGOFFICE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载